RESEARCH ARTICLE Open Access Transcriptional analysis of late ripening stages of grapevine berry Sabine Guillaumie 1,2† , Romain Fouquet 1,2† , Christian Kappel 1,2 , Céline Camps 1,2 , Nancy Terrier 3 , Dominique Moncomble 4 , Jake D Dunlevy 5 , Christopher Davies 6 , Paul K Boss 6 and Serge Delrot 1,2* Abstract Background: The composition of grapevine berry at harvest is a major determinant of wine quality. Optimal oenological maturity of berries is characterized by a high sugar/acidity ratio, high anthocyanin content in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition and berry tasting. In this context, it is interesting to identify genes that are expressed/repressed specifically at the late stages of ripening and which may be used as indicators of maturity. Results: Whole bunches and berries sorted by density were collected in vineyard on Chardonnay (white cultivar) grapevines for two consecutive years at three stages of ripening (7-days before harvest (TH-7), harvest (TH), and 10- days after harves t (TH+10)). Microvinification and sensory analysis indicate that the quality of the wines made from the whole bunches collected at TH-7, TH and TH+10 differed, TH providing the highest quality wines. In parallel, gene expression was studied with Qiagen/Operon microarrays using two types of samples, i.e. whole bunches and berries sorted by density. Only 12 genes were consistently up- or down-regulated in whole bunches and density sorted berries for the two years studied in Chardonnay. 52 genes were differentially expressed between the TH-7 and TH samples. In order to determine whether these genes followed a similar pattern of expression during the late stages of berry ripening in a red cultivar, nine genes were selected for RT-PCR analysis with Cabernet Sau vignon grown under two different temperature regimes affecting the precocity of ripening. The expression profiles and their relationship to ripening were confirmed in Cabernet Sauvignon for seven genes, encoding a carotenoid cleavage dioxygenase, a galactinol synthase, a late embryogenesis abundant protein, a dirigent-like protein, a histidine kinase receptor, a valencene synthase and a putative S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase. Conclusions: This set of up- and down-regulated genes characterize the late stages of berry ripening in the two cultivars studied, and are indirectly linked to wine quality. They might be used directly or indirectly to design immunological, biochemical or molecular tools aimed at the determination of optimal ripening in these cultivars. Background Grapevine (Vitis vinifera L.) is a nonclimacteric fruit species used as table fruit, dried raisins, and for vinifica- tion (wines) and distillation (liquors). In 2007, eight mil- lion hectares of grapevines produced 31 billion bottles of wine from vineyards throughout the world. Between 2003 and 2008, global consumption of wine has increased by 6% (International Organization of Vine and Wine (OIV) statistics). The composition of the grape berry at harvest is a major determinant of wine quality. It depends on the i nteracti ons between the genotypes of the rootstock and of the variety with the global environ- ment aro und the plant and the microenvironment around the berries. Grape development is divided into three phases i.e. two growth phases separated by a lag phase [1]. The first g rowth period, also called the herbaceous phase, is characterized by embryo development and cell divisions. During this phase, various solutes (malic and tartaric acids, tannins, hydroxycinnamic acids and aroma com- pounds) accumulate in the different tissues of the * Correspondence: serge.delrot@bordeaux.inra.fr † Contributed equally 1 Univ. Bordeaux, ISVV, Ecophysiologie et Génomique Fonctionnelle de la Vigne, UMR 1287, F-33140 Villenave d’Ornon, France Full list of author information is available at the end of the article Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 © 2011 Guillaumie et al; licensee BioMed Central Ltd. This is an Open Access articl e distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permi ts unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. berries [2]. All these compounds are important for wine quality. Tartaric and malic acids determine wine acidity , and hydroxycinnamic acids are precursors of phenolic volatiles. Tannins are responsible for the bitter and astringent taste of red wines. The phase called véraison is a transition phase charac- terized by a change of b erry skin color, from green to white or red, depending on the variety, by the beginning of berry softening, and by a sudde n increase in the rate of sugar accumulation. The end of véraison coincides with the onset of maturation, which represents the sec- ondperiodofberrygrowth,mainlyduetowaterinflux and cell enlargement. The maturation phase is charac- terized by dramatic changes in berry composition [2]. The concentration of some solutes (e.g. malic acid) which are accumulated during the first growth period, decline on a per-berry basis while the concentration s of other molecules (sugars, anthocyanins) strongly increase. Many aroma an d flavor compounds essential for wine typicity are produced at a late stage during grapevine ripening. Several relatively subjective definitions can be used to charact erize grape berry ripeness: physiological, techno- logical, aromatic , polyphenolic and oenological. Physio- logical ripeness corresponds to the time when the berry is ready to be disseminated for plant sexual reproduc- tion and propagation. Technological maturity is the timepointbeyondwhichberriesdonotaccumulate more sugars and do not lose any more acidity. Aromatic maturity is characterized by the optimal concentration of aroma and volatile compounds. Phenolic maturity takes into account the quantitative and qualitative evo- lution of the berry polyphenols in the skin (anthocya- nins and tannins) and seeds (tannins). However, none of them is really satisfactory because few biochemical markers are available, and ripeness depends on th eir combination and int eractions. The wine growers only consider the oenological maturity in order to determine the optimal date of harvest. The oenological maturity tries to take into account and opti- mize all the forms of maturity previously described while preserving the desired typicity of wines. Therefore, the grapevine berries harvested at oenological maturity show a high sugar/acidity ratio, high anthocyanin con- tent in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition (sugar and acid content, and total polyphenol) and on berry tasting. It is there- fore important to understand the physiological and molecular basis of grapevine berry ripening that may lead to oenological maturity. The availability of the grapevine genome [3,4] has boosted large-scale mRNA expression profiling studies of water and salinity stress [5], berry development and ripening [6-8], resistance against pathogenic fungi [9-11] or control of stilbene accumulation [12] using cDNA or oligonucleotide microarrays. Several multigenic famili es co ntrol the biosynthesis of molecules involved in the grape berry ripening. They are mostly related to cell-wall composition, sugar and water import, organic acid metabolism and storage, and flavo- noid synthesis [7,8]. One of the major difficulties cur- rently faced by the wine growers is the lack of accurate descriptors to predict the physiological state of berries. Even though some researchers have analyzed transcrip- tion changes during berry development and ripening [6-8], comprehensive transcript profil ing has never been used to investigate the last steps of grapevine ripening in relation to wine organoleptic properties. Thus, the signaling networks involved in regulation of the last stages of berry ripening are still unknown. The present study describes a detailed analysis of gene expression in Chardonnay berries sampled at three dif- ferent stages during late ripening. Biochemical analysis of grapevine berries and gustatory appraisals of microv i- nifications were also made. A limited set of genes were consistently differentially expressed in Chardonnay ber- ries whose different ripening stages resulted in different qualities of wine. The expression profiles of some of these genes were also studied and confirmed in the red cultivar Cabernet Sauvignon. The expression of these candidate genes is clearly altered during the last stages of ripening and thus may be considered as potential indicators of late ripening for both cultivars. Results and Disc ussion Characterization of Chardonnay samples The Vitis vinifera cv. Chardonnay berry samples were harvested over the course of berry ripening from the CIVC vineyard in Champagne (France) during fall 2005 and 2006. To take into account the heterogeneity of berry ripening in a vineyard, samples were harvested both as densimetrically sorted berries (DSB) and whole bunch berries (WBB) for better comparison. Samples were collected 7-days be fore harvest (TH-7), at theoreti- cal harvest (TH) and 10-days after harvest (TH+10). According to DSB, the most representative class was selected for the rest of the study and their density varied from 120 to 150 g/L NaCl (Figure 1). Berry weight, total soluble solids (°BRIX) and potential alcohol content of DSB harvested samples are given in Table 1. The evolu- tion of the mean berry weight of the major DSB class depends on the climate of the year. Mean berry weight remained constant in 2005, whereas it increased in 2006, particularly at the TH+10 stage. A ccording to the CIVC wine-making procedures, the technological matur- ity corresponded to i) berries free of disease, particularly free of gray mold (Botrytis cinerea) and powdery mildew Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 2 of 27 (Uncinula necator), ii) a potential alcohol content of 10.0% vol and iii) a total acid content of 8 g H 2 SO 4 /L. In practice, a potential alcohol content higher than 9.0% vol and lower than 12.0% vol (over-ripe) or a total acid content between 6 g H 2 SO 4 /L and 9 g H 2 SO 4 /L can express a high level of the qualitative potential in Cham- pagne wines. The combination and adjustment of the level of these thresholds to the highest quality of wines is ba sed on sensory analysis benchmarks. The potential alcohol and the total acid contents of Chardonnay har- vested samples from the CIV C vineyard during fall 2005 and 2006 ranged between 10.19 to 11.60% vol (Table 1) and 5.6 to 8.3 g H 2 SO 4 /L (Table 2) respectively. There- fore, TH-7, TH and TH+10 WBB and DSB samples cor- respondedtoanadequatetimespanforthestudyof ripening (Tables 1 and 2). Microvinification assays and sensory analysis Microvinification and sensory analyses were done to assess the quality of the wine produced from the berries harvested at the TH-7, TH and TH+10 stages. These analyses were performed to determine whether wines made from the TH-7, TH and TH+10 samples could be discriminated. The overall objective of these combined analyses was to show which harvest time point is the bestforproducingaqualityChampagnewinebetween the TH-7, TH and TH+10 harvest sta ges, and thus to associate a transcriptomic p rofile with the highest wine quality. The physicochemical parameters determined on the decanted must and base wines for Chardonnay wines are given in Table 2. Similar patterns for total sugar and alcohol contents were found in 2005 and 2006 for dec- antedmustwinesderivedfromTH-7,THandTH+10 samples. The same was true for the total acid contents in decanted must and base wines. During the ripening process, the sugar and alcohol contents increased in decanted must wines whereas the total acid contents decreased in decanted must and base wines. The sugar to acid ratio is not used in the Champagne area to determine the optimal harvest date, but it is commonly used as a quality index in grapevine [2]. The changes in total sugar/total acid ratio of the Chardonnay decanted musts during grapevine berry late ripening are therefor e Berry percentage A B Berry percentage B Berry percentage 80 90 100 110 120 130 140 150 160 80 90 100 110 120 130 140 150 160 Densit y classes (g /L NaCl ) Densit y classes (g /L NaCl ) TH-7 TH TH+10 Figure 1 Representativity of berry classes separated according to their density at three stages of Chardonnay ripening. In 2005 (A) and 2006 (B), one thousand berries were harvested at each of the three harvest date and were separated into classes according to their density. TH- 7, 7-days before theoretical harvest; TH, theoretical harvest; TH+10, 10-days after harvest. Table 1 Physiological characteristics of densimetrically sorted berries (DSB) of Vitis vinifera L. cv. Chardonnay grown in Epernay, France, in the 2005 and 2006 seasons, at three ripening stages Harvest date Density (g/L NaCl) Berry weight (g) Total soluble solids (°BRIX) Potential alcohol (% vol) 09/19/2005 (TH-7) 120 - 130 1.55 18.2 10.19 09/26/2005 (TH) 130 - 140 1.57 19.2 10.86 10/05/2005 (TH+10) 130 - 140 1.55 20.0 11.40 09/14/2006 (TH-7) 130 - 140 1.38 19.3 10.93 09/20/2006 (TH) 130 - 140 1.45 19.2 10.86 10/02/2006 (TH+10) 140 - 150 1.54 20.3 11.60 TH-7, 7-days before theoretical harvest; TH, theoretical harvest; TH+10, 10-days after harvest. Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 3 of 27 shown in Table 2. The total sugar/total acid ratio increased during the last stages of ripening process and ranged from 24.5 to 34.5 during fall 2005 and from 20 .8 to 31 during fall 2006. At the harvest stage (TH), the total sugar/total acid ratio was d ifferent between the decanted must wines de rived from samples harvested during fall 2005 and 2006 . They varie d from 29.5 (TH- 2005) to 24.6 (TH-2006). However, and if the 2005 and 2006 vintages ar e considered as repetitive, an average increase of 16.6% ± 1.6 of the sugar to acid ratio was observed between the TH-7 and TH musts. A similar pattern, i.e. an incre ase of 17.4% ± 4.2, was also noticed between the TH+10 and TH musts. Thus, a rise of 31.2% ± 2.2 was observed in the overall total sugar/total acid ratio between the TH-7 and TH+10 stages. In con- trast, total SO 2 , tartaric and L-malic acids, total nitro- gen, ammoniacal nitrogen and calcium contents in decanted must wines and potassium content in base wines showed different trends in 2005 and 2006 vin- tages. Among the amino acid contents, no difference and consistent evolution was noticed except for proline in decanted must wines of 2005 and 2006 vintages. Pro- line is not used by yeasts, but is classically high in the Chardonnay cultivar. Among all physicochemical parameters investigated in decanted must and base wines, the relative content in proline (% compared to all amino acids), the sugar, alcohol and total acid contents and consequently the sugar to acid ratio were the only parame ters displaying an evolution which can be related to the late ripening progress of Chardonnay berries. A sensory analysis was performed to distinguish the base wines elaborated with berries harvested at the TH- 7, TH and TH+10 stages (Table 3). For each growing season studied, a triangular test was conducted. The data did not reveal any significant difference between the base wines elaborated with the TH-7 and TH berries of the two growing seasons. However, the same compar- ison between TH and TH+10 or TH-7 and TH+10 base wines indicated significant variations for each year. Sen- sory analysis demonstrates that wines elaborated from the TH berry samples exhibit typical sensory properties of Champagne wines (Table 3). The TH-7 and TH+10 wines display aromas that are less typical or not typical at all. The global gene expression analysis in the different berry samples (i.e. WBB and DSB) and ripening stages provided us with a f ingerprint of the grap evine late ripening transcriptome. In this w ay, we identified ( 1) Table 2 Physicochemical parameters of microvinifications Harvest year 2005 2006 Wine stage DMU BW DMU BW Harvest date TH-7 TH TH +10 TH-7 TH TH +10 TH-7 TH TH +10 TH-7 TH TH +10 Total sugar content (densimetric titration, g/L) 170.0 183.0 193.0 - - - 173.0 180.0 192.0 - - - Alcohol content (densimetric titration, % vol) 10.1 10.9 11.4 - - - 10.3 10.7 11.4 - - - pH 3.0 3.1 2.9 3.1 3.1 3.2 2.9 3.1 3.1 3.1 3.1 3.2 Total acid content (potentiometric titration, g H 2 SO 4 / L) 7.0 6.2 5.6 5.0 4.6 4.1 8.3 7.3 6.2 5.1 4.7 4.1 Total enzymatic SO 2 content (Lisa method, mg/L) 13.0 29.0 29.0 33.0 37.0 49.0 34.0 20.0 29.0 37.0 37.0 49.0 Tartaric acid (g/L) 7.0 7.6 6.2 3.7 3.3 2.9 6.8 7.4 8.9 3.5 2.5 2.7 L-malic acid (g/L) 5.3 4.8 3.6 - - - 5.5 5.4 5.4 - - - Total sugar: total acid ratio 24.3 29.5 34.5 - - - 20.8 24.6 31.0 - - - Total nitrogen (mg N/L) 227.0 261.0 237.0 138.0 203.0 198.0 454.0 223.0 222.0 171.0 197.0 186.0 Ammoniacal nitrogen (Lisa method, mg N/L) 42.0 41.0 59.0 - - - 49.0 47.0 34.0 - - - Glucose + fructose (Lisa method, g/L) - - - 0.8 0.9 0.9 - - - 0.8 0.8 1.1 Abs atomic potassium (mg/L) 1310.0 1100.0 1480.0 565.0 477.0 484.0 1603.0 1563.0 1823.0 503.0 563.0 502.0 Abs atomic calcium (mg/L) 82.0 57.0 53.0 - - - 47.0 99.0 80.0 - - - Serine (%) 7.2 7.1 - - - - 9.8 6.8 - - - - Threonine (%) 4.5 4.1 - - - - 0.6 0.6 - - - - Asparagine (%) 0.9 1.0 - - - - 1.1 1.0 - - - - Glutamine (%) 11.5 8.2 - - - - 19.8 17.1 - - - - Proline (%) 28.3 36.7 - - - - 26.8 35.5 - - - - Alanine (%) 19.5 13.9 - - - - 17.8 16.0 - - - - y-aminobutyric acid (%) 6.4 5.0 - - - - 5.0 5.9 - - - - Arginine (%) 5.5 4.5 - - - - 9.0 7.6 - - - - Except proline which is not used by yeast, all amino acids noticed above correspond to 80% of yeast nitrogen needs. -, not tested; BW, base wine; DMU, decanted must; TH-7, 7-days before theoretical harvest; TH, theoretical harvest; TH+10, 10-days after harvest. Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 4 of 27 genes that are temporally co-expressed, and (2) indivi- dual gene family members that are preferentially expressed in a particular berry sample or ripening stage. Differential gene expression in Chardonnay berries during late ripening Transcriptomic analysis was conducted with the differ- ent berry samples (i.e. WBB and DSB). Samples col- lected at the TH stage were compared to the TH-7 and TH+10 stages respectively in order to emphasize evolu- tions of gene expression around the TH stage. Among the 14,562 investigated genes, 5 and 7 genes were consistently down-regulated or up-regulated throughout t he last steps of grapevine ripening in both WBB and DSB (Figure 2; Table 4) and for the two years studied. These genes belong to five functional categories, including aroma-, dessication- or pathogenesis-related genes and phenylpropanoid metabolism (Table 4). These putative functions were attributed on the basis o f homology with grape and Arabidopsis thaliana genes. The most homologous Arabidopsis thaliana and Vitis vinifera genes of each grape oligonucleotide are also indicated in Table 4. Among the 12 genes differentially expressed throughout the last phases of grapevine ripen- ing (TH-7, TH, TH+10) , three did not h ave any known function. Aroma related genes Aroma is important for wine quality, and it is therefore interesting that one gene predict ed to encode a putative carotenoid cleavage dioxygenase (CCD) was up- regulate d during Chardonnay ripening (Table 4 and Fig- ure 3). Indeed, carotenoids are apocarotenoid precursors which play a role in the production of phytohormones (i.e. abscisic acid) and some flavors and aromas. Apocar- otenoids are mos tly generated by the cleavage of a caro- tenoid molecule by en zymes of the CCD family [13-15]. Among the carotenoids, the levels of beta-carotene, lutein, flavoxanthin and neoxanthin decrease after vérai- son in grapevine berries [16]. These carotenoids undergo breakdown reactions that pr oduce C13 noriso- prenoid compounds involved in the typical aromas o f some grapevine cultivars [17] as was demonstrated with VvCCD1 [18]. The increased transcript abundance of VvCCD4a could be related to the presence of apocarote- noids during the end of the ripening process. In grape, four CCD4 genes have been identified in silico [19], but none has been functionally characte rized. According to Huang et al. [20], plants produce at least two different forms of CCD4 enzymes. Among the CCD4 proteins already characterized for other plants, the Malus domestica and Rosa x damas- cena CCD4 proteins (MdCCD4 and RdCCD4) are the closest to VvCCD4a. In vivo assays analyzed by SPME- GC-MS showed that MdCCD4 and RdCCD4 cleave b- carotene to y ield b-ionone [20]. However, no cleavage products were found when MdCCD4 and RdCCD4 genes were co-expressed in E. coli strains that accumu- lated linear carotenoids such as cis-z-carotene or lyco- pene [20]. We performed isolation and cloning of the VvCCD4a gene into pGEX expression vector. In a simi- lar way to various CCD4s such as MdCCD4 or RdCCD4 Table 3 Wine sensory analysis in Chardonnay base wines from 7-days before theoretical harvest (TH-7), theoretical harvest (TH) and 10-days after theoretical harvest (TH+10) samples of the 2005 and 2006 growing seasons Triangular tests Samples Major sensory descriptors 2005 TH-7/TH TH-7 Slight milk, lively TH Fruity (cherry), round, slight bitterness TH/TH+10* TH Milk (yoghurt, toffee, butter), round TH+10 Reductive character (sulfur), more vegetal than smoked and roasted, lively TH-7/TH+10* TH-7 Acid, lively, aggressive TH+10 Vegetal, less acid versus round and flat mouth, bitterness 2006 TH-7/TH TH-7 Acid (aggressive) TH Reductive hint (animal), less acid TH/TH+10* TH Reductive character (cauliflower), more acid than astringent and bitter TH+10 Reductive character (hydrocarbon, rubber, burnt wood, vegetable stock versus animal), acid (more aggressive), bitter, short TH-7/TH+10* TH-7 Reductive hint, acid (fresher, harder, aggressive), aqueous mouth TH+10 Roasted and reductive character (sulfur, animal, smoke, putrid), acid (hard, lively, slight acidity), round, bitter Tasting descriptors represent a summary of sensory descriptors employed by a tasting panel of 13 tasters to qualify Chardonnay base wines. *correspond to significant comparison analysis performed (P < 0.05). Bold sensory descriptors are the most representative ones distinguishing two defined base wines. Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 5 of 27 [20], the co-expression of VvCCD4a g ene in the strains accumulating cis-z-carotene, lycopene, b-carotene, and zeaxanthin did not cause a lack of pigmentation in these cultures (data no t shown). Although some CCD4 pro- teins have been shown to cleave carotenoid substrates at the 9,10 and 9’,10’ positions, they might have different biochemical functions as they may accept different (apo) carotenoids and show various expression profiles. A subcellular localization study of VvCCD4a protein revealed the c hloroplast localization of the VvCCD4a enzyme (data not shown). This is in agreement with the deduced amino acid sequences of all CCD4 proteins, including VvCCD4a, which contain a plastid-targeting transit peptide at the N-termin us [19]. Furthermore, the Crocus sativus an d Arabidopsis CCD4s, have been shown to reside in plastids, where their substrates are localized , suggesting a direct involvement in volatile for- mation [19]. Altogether, this suggests a potential role for VvCCD4a in berry color, flavor and a roma during late ripening of Chardonnay berries. Phenylpropanoid pathway Two genes called VvPAL1 and VvPAL2 encoding pheny- lalanine ammoni a-lyase (PAL; EC 4.3.1.5) were up-regu- lated throughout the last periods of Chardonnay ripening (Table 4). PAL catalyzes the first step in the phenylpropanoid pathway by removing the NH3 radical from L-Phe to produce trans-cinnamic acid and other phenolic compounds. In grapevine berry, PAL is located in epidermal cells as we ll as in the seeds [reviewed in [21]]. PAL acti vity within the grapevine skin is maximal at the first stages of development, and decreases up to véraison. In colored grapevines, PAL activity in the skin WBB TH vs TH-7 WBB TH+10 vs TH DSB TH vs TH-7 DSB TH+10 vs TH p- value<0.05 37 148 108 12 52 53 23 97 167 Figure 2 Venn diagram summary of differentially expressed genes identified in Chardonnay at three stages of ripening. Chardonnay whole bunches and densimetrically sorted berries were harvested at the 7-days before harvest (TH-7), theoretical harvest (TH) and 10-days after harvest (TH+10) stages during the 2005 and 2006 years. Comparisons of the expression profiles of TH versus TH-7 and TH+10 versus TH were made for whole bunches and densimetrically sorted berries. The total numbers of genes differentially expressed are indicated in respective circles (P < 0.05, ≥1.75-fold). The combined number of genes simultaneously up- or down-regulated is given in intersections between circles. Twelve genes were differentially expressed at all stages of late ripening; some genes were stage or sample type specific while others were overlapping in two stage or sample comparisons (for gene identity, see Tables 4, 5 and 6 and Additional files 1 and 2(Tables S1and S2)). WBB, whole bunch berries; DSB, densimetrically sorted berries. Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 6 of 27 Table 4 Differentially expressed genes (P < 0.05, ≥1.75-fold) in Chardonnay grapevine berries all along the investigated ripening periods of the 2005 and 2006 growing seasons Putative function Grape Microarray Accession Number (Vv_#) Grape Nucleotide Accession Number (mRNA) Grape Gene Accession Number (GSVIVT#) Most Homologous Arabidopsis Sequence WBB average ratio (TH vs TH-7) p-value WBB average ratio (TH+10 vs TH) p-value DSB average ratio (TH vs TH-7) p-value DSB average ratio (TH+10 vs TH) p-value Aroma related genes Carotenoid cleavage dioxygenase 4a (VvCCD4a) Vv_10003015 XM_002268368 GSVIVT01036862001 At4g19170 0.999 2.00E-04 1.219 9.00E-05 1.071 0.00041 1.018 0.00022 Phenylpropanoid/ lignin genes Phenylalanine ammonia lyase (VvPAL1) Vv_10000977 XM_002281763 GSVIVT01025703001 At2g37040 1.691 6.00E-05 1.076 7.00E-04 1.052 0.00107 1.497 0.00031 Phenylalanine ammonia lyase (VvPAL2) Vv_10000978 AB015871 GSVIVT01024306001 At3g53260 1.858 0.00013 1.046 0.00069 1.234 0.00025 1.578 0.00024 Response to dessication Galactinol synthase (VvGolS) Vv_10000327 XM_002262669 GSVIVT01017634001 At1g56600 -1.307 0.00057 -0.99 0.00142 -1.462 0.00057 -1.107 0.00053 Late embryogenesis abundant protein (VvLEA1) Vv_10001081 XM_002283966 GSVIVT01033739001 At3g53040 -1.11 3.00E-04 -1.193 0.00118 -1.378 2.00E-04 -1.071 0.00041 Late embryogenesis abundant protein Vv_10001082 AM474201 GSVIVT01033739001 At3g53040 -1.271 0.00053 -1.3 0.00017 -1.559 0.00016 -1.27 1.00E-04 Pathogenesis- related genes Pathogenesis- related protein 10 Vv_10010887 XM_002274581 GSVIVT01035059001 - 1.957 0.00178 2.077 0.0022 1.375 0.00037 2.115 0.00055 Dirigent-like protein (VvDIR-like) Vv_10002588 XM_002285641 GSVIVT01025392001 At3g13650 1.979 0.00087 1.81 0.00053 1.377 0.00456 1.734 4.00E-04 Hormonal control Histidine kinase receptor (VvHKR) Vv_10014467 FJ822975 GSVIVT01030060001 At5g35750 -0.943 0.00023 -0.845 0.00031 -0.826 0.00033 -0.94 0.00032 Unknown function Unknown gene Vv_10014451 XM_002270095 GSVIVT01010993001 At4g25010 2.263 3.00E-05 0.982 0.00021 1.757 3.00E-05 1.471 6.00E-05 Unknown gene Vv_10002806 XM_002273032 GSVIVT01038103001 At1g65260 1.375 0.00014 0.884 0.00106 1.01 0.00122 1.095 0.00058 Unknown gene Vv_10011055 XM_002284158 - - -0.849 0.00238 -0.811 0.00091 -0.838 0.00113 -1.032 0.00115 Genes are organized in functional categories. Ratio values are presented as log2. DSB, densimetrically sorted berries; TH-7, 7-days before theoretical harvest; TH, theoretical harvest; TH+10, 10-days after harvest; WBB, whole bunch berries. Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 7 of 27 shows a second peak after véraison [reviewed in [21]]. There is a close relationship between its activity and the color intensity of colored grapevines [22]. No PAL iso- enzyme is detected in the skin of n on-colored berries such as Chardonnay during th e late ripening [23], nor is the re any PAL transcript present [24]. Thus, PAL activ- ity seems to play an essential role in anthocyanin accu- mulation only in colored grapevine berries. To date, the putative functions of VvPAL1 and VvPAL2 in ripening of white grapevine berries are still unknown. In Arabi- dopsis thaliana, AtPAL 1 and AtPAL2 are related to the lignification process [25]. AtPAL1 and AtPAL2 are responsive to environmental fact ors like nitrogen deple- tion or pathogens [26]. Such roles can also be hypothe- sized for VvPAL1 and VvPAL2. Response to dessication Galactinol synthase (GolS; EC 2.4.1.12 3) is a member of the glycosyl transferase family 8 (GT8) [27] and cata- lyzes the first committed step in the biosynthesis path- way of raffinose family oligosaccharides (RFOs). GolS synthesizes galactinol, whichservesasadonortoform soluble galactosyl-Suc carbohydrates. Accumulation of RFOs is usually associated with abiotic stress such as cold, heat or dehydration [28]. At the p rotein level, VvGolS (GSVIVP00019670001; Ta ble 4) exh ibited 69% identity/78% simil arity with Arabidopsis GolS1. AtGolS1 transcripts were detected during seed maturation and may be implicated in seed osm oprotection [29]. How- ever, RFOs a lso constitute a significant component of phloem-transported sugars in some plants [30]. Two genes encoding late embryogenesis abundant proteins (LEA) were also down-regu lated during the last stages of grape ripening (Table 4). LEA expression could be related to the acquisition of dessication tolerance in seeds; but many LEA proteins are induced by cold, osmotic stress or exogenous abscisic acid, or can even be expressed constitutively [31]. Pathogenesis-related genes PR proteins are induced in response to several pathogen agents (bacteria, viruses and fungi) during the hypersen- sitive response (HR) and systemic acquired resistance (SAR) [32]. The PR proteins form a heterogeneous family including 17 groups (PR-1 to PR-17) distin- guished on the basis of structural homologies [ 32,33]. However, the biological and biochemical functions of these proteins during the defense reactions and develop- mental processes are still unclear. The pathogenesis-related proteins (PR) c omprise the vast majority of wine proteins and adversely affect the clarity and st ability of wines [34]. The expression of one gene encod ing a PR-10 prot ein was up-regulate d during the later stages of grapevine ripening, and especially at theTH+10stage(Table4).Ingeneral,PR-10proteins exhibit allergenic, anti-fungal and ribonuclease activities. Robert et al. [35] emphasized the accumulation of PR- 10 proteins in grapevine after Pseudomonas syringae infection, which was ascribed to HR. Up-regulation of PR-10 expression may be due to attacks of Botrytis cinerea which occurred in Champagne vineyards during the last stages of ripening in 2005 and 2006. 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 2005 2006 VvCCD4a relative expression Figure 3 Bar diagram of Vi tis vinifera carotenoid cleavage dioxygenase 4a (VvCCD4a) transcript abundance: a comparison of qRT-PCR data of Chardonnay whole bunch and densimetrically sorted berries harvested at three ripening stages in 2005 and 2006. The mRNA level was expressed relative to controls (set at 1), reference gene EF1-a. RT-PCR data are reported as means ± SE (error bars) of n = 3 technical replicates. DSB, densimetrically sorted berries; TH-7, 7-days before theoretical harvest; TH, theoretical harvest; TH+10, 10-days after harvest; WBB, whole bunch berries. Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 8 of 27 In addition, one gene encoding a putative dirigent-like protein (DIR-like) was up-regulated during the berry late ripening (Table 4). This gene displays sequence homol- ogy to members of the DIR-b subfamily i.e. PDIR3, PDIR7 and PDIR20 of Picea glauca × engelmanni, Picea glauca and Picea sitchensis respectively [36]. The ability of DIR proteins to direct the stereoselective formation of lignans has been previously demonstrated with in vitro assays for several members of the DIR-a subfamily from Forsythia intermedia [37]. However, the biochemical functions for the members of DIR-b, DIR-c, DIR-d a nd DIR-e subfamilies are not known so that the members of these subfamilies are referred to as DIR-like. In Sitka spruce trees, the expression of several DIR genes was altered by biotic and abiotic stresses, suggesting their implication in plant defense [37]. Hormonal control In the present study, only one gene (FJ822975, termed as VvCyt1) encoding a cytokinin histidine-kinase receptor, related to hormone metabolism and regulation of berry development and ripening, was down-regulated through- out the last steps of ripening (Table 4). The ARABIDOPSIS HISTIDINE KINASE 2 (AHK2) gene is the closest homolo- gue to VvCyt1. Cytokinins regulate the development of vascular bundles in inflorescence stems of Arabidopsis thaliana via the AHK2 signaling pathway [38]. Cytokinin activity is significant duri ng the early stages of grapevine berry development but decreases later on during ripening [2]. To date, the putative functions of VvCyt1 during the grapevine berry ripening remain to be clarified. In summary, a total of 12 genes have been shown to be consistently regulated throughout the last steps of the ripening process and can be co nsidered as new indi- cators of late ripening in Chardonnay. With regard to the five down-regulated genes, an average down-regula- tion of 2-fold was observed between the TH and TH -7 samples and also between the TH+10 and TH ones. Similarly, an average up-regulation of 3.4-fold and 2.6- fold was observed i n the TH versus TH-7 and in TH +10 versus TH samples respectively. These average expression ratios could be related not only to the increase of the sugar to acid ratio (Table 2) throughout the last steps of grapevine ripening but also to the sen- sory analysis performed (Table 3). The formati on of fla- vors in the ripening grape berry results from the balance of the sugar to acid ratio as well as synthesis of flavor and aromatic compounds [2]. The present study links the sugar to acid ratio, the sensory characteristics and the expression profiles of some specific genes. TH versus TH-7 differential gene expression in Chardonnay berries This comparison allows genes that are differentially expressed just before technological maturity to be identified. Among the genes expressed at the TH-7 and TH stages in WBB and DSB, 52 genes were differentially regulated in TH versus TH-7 berries. In addition to the 12 previously mentioned as up- or down-regulated throughout all the stages of ripening process, 20 more genes associated to a putative function were differen- tially expressed in TH versus TH-7 WBB and DSB sam- ples (Table 5). Genes representing hypothetical proteins of unknown function are shown in Additional file 1 (Table S1). Cell wall-related genes Fruit development and ripening involve the action of a complex set of enzymes and proteins associated with the disassembly of primary cell wall and reduction in cell-ce ll adhesion [39]. The expansins, xyloglucan endo- transglycosylases/hydrolases and galacturonosyltrans- ferases belong to this set of enzymes. The expansins are able to plasticize the cellulose- hemicellulose network of plant cell wall. In the litera- ture, three putative EXP genes, Vlexp1, Vlexp2,and Vlexp3 have been isolated from Kyoho grape (Vitis lab- rusca x Vitis vi nife ra) berries and their expression was monitoredatninestagesofberrydevelopment[40]. Vlexp1 is the closest homologue to the grapevine EXPA gene (GSVIVT01007 987001), which is differentially expressed between the TH-7 and TH stages (Table 5). Vlexp1 expression increased with berry development up to the half-colored stage and then decreased during the later stages of maturation [40]. In strawberry, FaEXPA4 (DQ183068) is the closest homologue of VvEXPA.At the protein level, VvEXPA (Table 5) exhibited 79% iden- tity/88% similarity with FaEXPA4. FaEXP4 mRNA is strongly expressed throughout fruit development and ripening, and exhibits a slight decrease at the end of maturity in Selva fruits, the firmest cultivar considered in the study of Dotto et al. [41]. This suggests that VvEXPA could be associated with the cell expansion and grapevine berry ripening (Table 5). Changes in the pectin matrix are regarded as an important factor that aff ects the cell wall structure dur- ing the fruit ripening and senescence [42]. a-(1,4)- Galacturonosyltransferases catalyze the addition of (1,4)- linked a-D-galacturonosyl residues onto the nonredu- cing end of homogalacturonan chains [43]. One g ene encoding such a putative galacturonosyltransferase was down-regulated at the TH stage in comparison with the TH-7 one. Xyloglucan is the principal hemicellulose component in the primary cell w alls of non-graminaceous plants, and accounts for 10% of the cell wall composition in grapevine berries [44] . During the fruit ripening process, xyloglucan degradation is the terminal cell wall degrada- tion that occurs [45]. Xyloglucan endotransglycosyl ases/ hydrolases (XTH) are involved in splitting and/or Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 9 of 27 Table 5 Differentially expressed genes (P < 0.05, ≥1.75-fold) in Chardonnay grapevine berries between theoretical harvest date (TH) and one week before (TH-7) of the 2005 and 2006 growing seasons. Putative function Grape Microarray Accession Number (Vv_#) Grape Nucleotide Accession Number (mRNA) Grape Gene Accession Number (GSVIVT#) Most Homologous Arabidopsis Sequence WBB average ratio p-value DSB average ratio p-value Cell wall related genes Alpha-expansin Vv_10001623 XM_002284822 GSVIVT01007987001 At1g69530 -1.14 0.00032 -0.829 0.00246 Polygalacturonate 4-alpha- galacturonosyltransferase Vv_10003714 XM_002271124 GSVIVT01020141001 At1g70090 -1.501 0.00027 -1.585 0.00022 Xyloglucan endotransglycosylase/hydrolase Vv_10011203 XM_002274118 GSVIVT01029170001 At5g57550 -0.884 0.00045 -0.799 0.00062 Xyloglucan endotransglycosylase/hydrolase Vv_10011021 XM_002274791 GSVIVT01029162001 At5g57560 -1.223 0.00014 -1.2 0.00018 Xyloglucan endotransglycosylase/hydrolase Vv_10010901 XM_002262725 GSVIVT01031601001 At3g23730 -1.1 0.00033 -1.36 0.00014 Xyloglucan endotransglucosylase/ hydrolase Vv_10011290 XM_002274516 GSVIVT01029166001 At4g25810 -1.472 6.00E-05 -1.475 7.00E-05 Biotic and abiotic stress related proteins Pathogenesis-related protein 10 Vv_10003874 XM_002274749 GSVIVT01035055001 - 1.542 0.00156 1.086 0.00016 Miraculin-like protein Vv_10011266 XM_002266394 GSVIVT01012922001 At1g17860 -2.409 9.00E-05 -0.994 0.00104 Transporters Sulfate transporter Vv_10001315 XM_002279177 GSVIVT01018028001 At3g51895 -1.207 0.00084 -0.907 0.00169 Transcription factors TCP transcription factor Vv_10010249 XM_002272192 GSVIVT01012766001 At1g72010 -1.233 0.00021 -1.038 0.00014 bZIP transcription factor Vv_10007432 XM_002285275 GSVIVT01014246001 At3g58120 -1.499 7.00E-05 -1.535 5.00E-05 Miscellaneous Phosphate-induced protein Vv_10000589 XM_002285726 GSVIVT01009065001 At4g08950 -2.342 0.00021 -1.885 2.00E-04 Phosphate-induced protein Vv_10000871 XM_002282859 GSVIVT01023873001 At2g17230 -1.141 0.00157 -1.184 0.00098 beta-ketoacyl-CoA synthase Vv_10004485 XM_002284950 GSVIVT01015472001 At2g26640 -0.845 0.00211 -0.883 0.00212 Metal ion binding protein Vv_10004892 XM_002281195 GSVIVT01022185001 At4g39700 -1.045 0.00016 -1.325 0.00011 AAA-type ATPase family protein Vv_10010867 XM_002268820 GSVIVT01023336001 At3g28600 -1.28 0.00026 -1.176 0.00015 AAA-type ATPase family protein Vv_10012487 XM_002280929 GSVIVT01015385001 At3g24530 -0.805 0.00072 -0.859 0.00099 Aspartyl protease protein Vv_10002995 XM_002265735 GSVIVT01036694001 At3g12700 -2.0 6.00E-05 -1.975 3.00E-05 Protease inhibitor Vv_10001691 XM_002266266 GSVIVT01012936001 At1g17860 -3.046 1.00E-05 -0.872 0.00418 PS60 protein/multicopper oxidase Vv_10000492 XM_002282178 GSVIVT01023902001 At1g76160 -0.922 0.00088 -0.846 0.00141 Genes are organized in functional categories. Ratio values are presented as log2. DSB, densimetrically sorted berries; TH-7, 7-days before theoretical harvest; TH, theoretical harvest; WBB, whole bunch berries. Guillaumie et al. BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 10 of 27 [...]... changes which alters grapevine physiology, berry growth ripening and content, the identification of genes linked to the late stages of grapevine ripening is important Conclusions The last steps of grapevine ripening involve a correlative differential expression of numerous genes However, based on the comparison of whole bunches vs densimetrically sorted berries, of two vintages, and of two cultivars (white... abundance of the VvValCS gene is likely an indicator for the synthesis of some aroma-related compounds at the latest stages of the ripening process Deluc et al [62] investigated the expression profile of the VvValCS gene during grapevine berry development under normal and water stress culture conditions in Chardonnay and Cabernet Sauvignon cultivars At the end of ripening, the VvValCS transcript profile... up-regulated throughout the last phases of Chardonnay ripening (TH-7, TH, TH+10) Similarly VvGolS, VvLEA1 and VvHKR were down-regulated throughout the same last phases of Chardonnay ripening The miraculin-like gene was down-regulated in TH versus TH-7 berries and VvValCS was a member of the 53 genes differentially regulated in TH+10 versus TH berries First of all the general aspects of expression profiles... on the influence of temperature on grapevine berry development especially on ripening process [92] Gene expression analysis was performed on Cabernet Sauvignon berries harvested from 7 to 19 weeks postflowering i.e from vộraison of 30C days and 25C nights berries to harvest of berries exposed to 20C days and 15C nights The expression profiles of nine candidate genes of Chardonnay late ripening were quantified... Page 27 of 27 gene expression during grape berry (Vitis vinifera L.) development Planta 2005, 222:832-847 99 Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods 2001, 25:402-408 doi:10.1186/1471-2229-11-165 Cite this article as: Guillaumie et al.: Transcriptional analysis of late ripening stages of grapevine. .. steps of berry ripening have never been used together to investigate the last stages of grapevine ripening Moreover, the intervals used here between two sampling times (seven to ten days) are much shorter than those used in previous studies [6-8] Is there an indicator of ripening status spreading during Chardonnay berry ripening? At the TH stage in comparison with the TH-7 stage, the transcript level of. .. berries Page 17 of 27 Guillaumie et al BMC Plant Biology 2011, 11:165 http://www.biomedcentral.com/1471-2229/11/165 Page 18 of 27 A Validation of some Chardonnay ripening indicators within another cultivar C Percent of colored berries B BRIX It is interesting and important to determine whether the genes which are consistently affected during the late stages of Chardonnay (white cultivar) ripening in Champagne... the restructuring of the cellulose-xyloglucan-pectin network during the fruit ripening [45] Plant defense proteins Another PR-10 gene is up-regulated during the grapevine ripening, especially between TH-7 and TH stages Among the stress-related genes, one gene homologous to a miraculin-like protein is also down-regulated (Table 5) A miraculin is a plant protein purified from extracts of miracle fruit... http://www.biomedcentral.com/1471-2229/11/165 genes The onset of mango softening and ripening is closely related to an increase in the MiPel PL gene expression, PL activity and pectin solubilization [79] Taken together, these data suggest a crucial role of VvPL1 gene during the berry ripening of white grapevine cultivars Hormone metabolism and regulation Among the hormone-related genes, only two genes associated with... of inorganic ions, sugars and organic acids In grape, Terrier et al [87] and Venter et al [88] already identified and characterized two isoforms of the V-PPase gene named VVPP1 and vpp2 respectively During the grapevine berry ripening, the V-PPase Page 16 of 27 activity apparently increases in parallel with the transcript levels of vpp2 and VVPP1 [87,88] The expression pattern of Vpp2 is also modulated . RESEARCH ARTICLE Open Access Transcriptional analysis of late ripening stages of grapevine berry Sabine Guillaumie 1,2† , Romain Fouquet 1,2† , Christian. regulation of the last stages of berry ripening are still unknown. The present study describes a detailed analysis of gene expression in Chardonnay berries sampled at three dif- ferent stages during late. Sauvignon. The expression of these candidate genes is clearly altered during the last stages of ripening and thus may be considered as potential indicators of late ripening for both cultivars. Results